Mild Drought Stress‐Induced Changes in Yield,Physiological Processes and Chemical Composition in Festuca,Lolium and Festulolium

The impact of mild drought stress (3 weeks at 40 % field water capacity) on yield, physiological processes, accumulation of proline and phenolic compounds and forage quality parameters in forage grasses was evaluated in pot experiments. During four different growing periods, the effects of water deficit were assessed in nine varieties from five species (Lolium perenne, Lolium multiflorum, Festuca pratensis, Festuca arundinacea and Festulolium braunii). All measured parameters were affected by drought stress in the different cuts. Photosynthesis, transpiration rate, stomatal conductance and dry matter yield were significantly lower in drought stress than under well‐watered conditions in all varieties. Higher water‐use efficiency was only observed during the first and fourth drought period, while this was not the case in the second and third. Mild drought stress significantly increased the content of proline, phenolic acid, flavonoids, water‐soluble carbohydrates and protein. All tested grasses showed also an increase of organic matter digestibility and cell wall digestibility under drought stress conditions.     

Intergeneric symmetric and asymmetric somatic hybridization in Festuca and Lolium

Summary Intergeneric symmetric and asymmetric somatic hybrids have been obtained by fusion of metabolically inactivated protoplasts from embryogenic suspension cultures of tall fescue (Festuca arundinacea Schreb.) and unirradiated or 10–500 Gy-irradiated protoplasts from non-morphogenic cell suspensions of Italian ryegrass (Lolium multiflorum Lam.). Genotypically and phenotypically different somatic hybrid Festulolium mature flowering plants were regenerated.Species-specific sequences from F. arundinacea and L. multiflorum being dispersed and evenly-represented in the corresponding genomes were isolated and used for the molecular characterization of the nuclear make-up of the intergeneric, somatic Festulolium plants recovered. The irradiation of Italian ryegrass protoplasts with 250 Gy X-rays prior to fusogenic treatment favoured the unidirectional elimination of most or part of the donor chromosomes. Irradiation of L. multiflorum protoplasts with 500 Gy produced highly asymmetric (over 80% donor genome elimination) nuclear hybrids and clones showing a complete loss of donor chromosomes.The RFLP analysis of the organellar composition in symmetric and asymmetric tall fescue (+) Italian ryegrass regenerants confirmed their somatic hybrid character and revealed a bias towards recipient-type organelles when extensive donor nuclear genome elimination had occurred.Approaches aimed at improving persistence of ryegrasses based on asymmetric somatic hybridization with largely sexually-incompatible grass species (F. rubra and Alopecurus pratensis), and at transferring the cytoplasmic male sterility trait by intra- and inter-specific hybridization in L. multiflorum and L. perenne, have been undertaken.Abbreviations cpDNA chloroplast DNA - CMS cytoplasmic male sterility - 2,4-D 2,4-dichlorophenoxy-acetic acid - IOA iodoacetamide - mtDNA mitochondrial DNA - RFLP restriction fragment length polymorphism     

Androgenesis from a Lolium multiflorum × Festuca arundinacea hybrid to generate extreme variation for freezing-tolerance

New opportunities for plant breeding using androgenesis in Lolium × Festuca hybrids have been identified. Plants derived by anther culture from a Lolium multiflorum × Festuca arundinacea (5x) hybrid were screened for freezing-tolerance, and their post-freezing recovery compared. The androgenic population showed extreme diversity in freezing-tolerance. While the majority of androgenic plants had inferior freezing-tolerance compared with the freezing-sensitive L. multiflorum parent, 6% of the population were more freezing resistant than the freezing-tolerant Festuca parent. Novel Lolium and Festuca gene combinations resulting from rare meiotic events were recovered within the androgenic population. The two most freezing-tolerant androgenic plants carried virtually the entire F. pratensis subgenome of F. arundinacea. F. pratensis is known to carry genes for freezing-tolerance and would be expected to be the primary source of genes governing this trait within the F. arundinacea genome. The most freezing-tolerant androgenic plants were more freezing-tolerant than the hybrid plant from which they were derived. Consequently, androgenesis was also effective in removing factor(s) reducing the expression of freezing-tolerance within the L. multiflorum × F. arundinacea (5x) hybrid.     

Temperature‐Dependent Growth Adaptation of Festuca pratensis from the Biophysical Viewpoint

Meadow fescue (Festuca pratensis), Italian ryegrass (Lolium multiflorum), their hybrid Festulolium braunii and perennial ryegrass (Lolium perenne) were grown hydroponically under vegetative (20 °C) and hardening (8 °C, 0 °C) regimes. The relative shoot/root growth ratio K, linear root growth rate and other parameters were estimated. When the temperature was lowered from 20 °C to 8 °C, the relative shoot/root growth ratio K of F. pratensis decreased to 0.6, i.e. much more than that of Lolium, suggesting that at 8 °C F. pratensis shoot growth blockage occurs. Further, by dropping the temperature from 8 °C down to 0 °C, the K ratio of F. pratensis increased significantly, while the linear root growth rate decreased much more markedly than in the other species – it suggests also a blockage of root growth. This growth slowdown of stress‐tolerant F. pratensis shoots and at a lower temperature also of its roots is inadequate to the direct impact of temperature and, thus, indicates a two‐step qualitative reorganization, i.e. transition into a qualitatively new state of stress. This phenomenon does not manifest itself in the less stress‐tolerant Lolium and Festulolium species but is expressed in stress‐tolerators, allowing them to achieve their strategic goal – to survive under extreme conditions.     

Culture of embryos and shoot tips for chromosome doubling in Lolium perenne and sterile hybrids between Lolium and Festuca

An improved method is reported for polyploid induction in Lolium (ryegrass), and in sterile F₁ hybrids between Lolium and Festuca (fescue). Two factors greatly increased the survival rate of colchicine‐treated embryos of Lolium perenne (perennial ryegrass) germinated and cultured in vitro (1) a high concentration of sucrose (100 g/1) in the germination medium and (1), maintenance at a low temperature of 10°C for 2 weeks after treatment. The maximum success rate for chromosome doubling among survivors of perennial ryegrass was 79.1%, and for L. perenne×Festuca arundinacea F₁ hybrid embryos it exceeded 40%. The same doubling treatment also works with shoot tip culture in non‐ flowering genotypes obtained by anther culture of L. multiflorum×F. arundinacea hybrids.     

Unreduced gamete in interspecific hybrids in the Festuca/Lolium complex

Self-sterile interspecific hybrids between diploid Lolium perenne and either Festuca arundinacea or F. gigantea (both hexaploids) set some seeds when subjected to a heavy pollen cloud from the amphiploids derived from the hybrids. All the progeny were octoploid and were formed from the fertilization of unreduced gametes in the F₁ hybrid by amphiploid pollen. The significance of the results in relation to breeding efforts within the Lolium/Festuca complex is discussed.     

Photosynthetic Apparatus Responses to Short‐term low‐temperature Flooding May Contribute to Freezing Tolerance Changes in Forage Grasses

Winter conditions are subjected to rapid climate changes. Increased precipitation and snow melting during warmer winters may result in low‐temperature flooding. These factors probably affect plant overwintering strategies. This study investigated the relationships between the photosynthetic adjustment and freezing tolerance following a short‐term low‐temperature flooding in two forage grasses (Lolium perenne and Festuca pratensis). The effect of flooding on the photosynthetic apparatus acclimation to cold was determined using chlorophyll a fluorescence measurements. Freezing tolerance was estimated using an electrolyte leakage test. It has been shown that genotypes activating non‐photochemical mechanisms of photosynthetic acclimation to cold during low‐temperature flooding may show increased freezing tolerance. Freezing tolerance reduction observed in Festuca pratensis was probably connected with decreased photochemical activity and photoinhibition of photosynthesis. It can be concluded that photosynthetic apparatus response to low‐temperature flooding may contribute to changes in the freezing tolerance. The direction of the changes is associated with different photosynthetic apparatus performance.     

Genomic constitution of Festulolium cultivars released in the Czech Republic

Genomic in situ hybridization (GISH) was used to characterize the chromosome constitutions of individual plants from a set of tetraploid and hexaploid cultivars of Festulolium developed and released in the Czech Republic from hybrids of Lolium multiflorum with Festuca pratensis and F. arundinacea. A simplified GISH protocol readily discriminated parental genomes in the hybrids and facilitated the screening of large numbers of plants per accession. The contribution of parental genomes in the cultivars tested ranged from predominance of chromatin from one of the parents to a more balanced contribution from both parents. However, in none of the cultivars were equal proportions of chromatin from both parents present. The parental contribution to the hybrids was both in the form of complete chromosomes or as chromosome translocations. In hexaploid cultivars from (L. multiflorum × F. arundinacea) × F. arundinacea hybrids the average numbers of complete L. multiflorum chromosomes ranged from 4.95 to 7.5 and the numbers of translocations from 6.33 to 10.21. Two tetraploid cultivars from (L. multiflorum × F. arundinacea) × L. multiflorum hybrids showed a strong prevalence of L. multiflorum chromatin and intergeneric translocations were rare. In the tetraploid cultivar ‘Perun’ of the L. multiflorum × F. pratensis hybrid there were 11.7 chromosomes of L. multiflorum and 14.7 recombined chromosomes on average. Reasons for the domination of one of the parental genomes in hybrid cultivars are not clear and are only partially explained by breeding history. Recombination rates of individual genomes in hybrids involving F. arundinacea were evaluated in double hybridization experiments. The results indicated a strong affinity of the L. multiflorum genome for the F. pratensis genome present in F. arundinacea and little affinity for the F. glaucescens genome. This suggests that introgressions from F. arundinacea into L. multiflorum are primarily limited to the F. pratensis genome which can be more readily accessed in L. multiflorum × F. pratensis hybrids.     

First in vivo assessment of feeding value of Festulolium hybrids derived from Festuca arundinacea var. glaucescens and selection for palatability

This paper reports on the first results of feeding value experiments, assessed with sheep, of hybrids between tetraploid Italian ryegrass (Lolium multiflorum, 2n = 4×= 28) and a progenitor of tall fescue, Festuca arundinacea var. glaucescens (2n = 4×= 28). Three hybrid populations resulting from either high, low or no selection for palatability were compared with a variety of Italian ryegrass as control and two varieties of the cultivated hexaploid (2n = 6×= 42) tall fescue, one of which had been selected for high palatability. On average, the hybrid populations were found to be as palatable as the improved tall fescue with voluntary intake (VI), in vivo digestibility of organic matter (DOM), and net energy expressed in fodder units for milk, similar to the best palatable tall fescue and to Italian ryegrass. However, selection for improved palatability among hybrids did not result in any further improvement of other traits, except VI in the hybrid selected for high palatability which significantly exceeded VI of Italian ryegrass. In conclusion, intergeneric hybridization between L. multiflorum and F. arundinacea var. glaucescens led to an immediate increase of feeding value compared with selection for palatability in tall fescue. However, to improve feeding value of hybrids further, similar selection for high palatability appeared questionable and possibly detrimental for other agronomic traits because of complicated inheritance in tetraploid L. multiflorum×F. glaucescens hybrids.     

Preliminary evaluation of turfgrass (perennial ryegrass and red fescue) cultivars for resistance to red thread disease using artificial infection

Selection of main cool‐season turfgrasses (perennial ryegrass and red fescue) for resistance to red thread disease caused by Laetisaria fuciformis has not progressed during the last 30 years because of low reproducibility of experiments carried out under natural infection. In this study, evaluation of a set of the turfgrass cultivars, including non‐host fodder grasses as control, was performed in a climatic chamber, a cold house and in the field using three types of artificial inoculum, and correlations between trials were analysed. In general, cultivars of Festuca rubra were more susceptible to the disease but differentiated better than cultivars of Lolium perenne. Significant interactions between cultivars of L. perenne and F. rubra and isolates of L. fuciformis were found. The fungus was also able to colonize leaves of sow thistle and wheat seedlings. In field conditions and in the glass cold‐house Dactylis glomerata was found to be relatively resistant to red thread disease. A possible selection programme is proposed.     

The Effect of Sward Management on the Mineral Content of Winter Grazed Herbage

No information is available regarding the mineral content of autumn‐saved herbage during winter grazing under Central European conditions. Therefore, P, K, Na, Mg and Ca concentrations of autumn‐saved growths of Festuca arundinacea and Lolium perenne were analysed considering the potential influence of pre‐utilization (saved from June or July), date of winter harvest (December, January, February) and year (three winters). For all years date of winter harvest was the main source of variance, whereas date of preceding cut had no relevant effect on the mineral concentrations during winter. Already in December P [2.3 to 4.0 g kg^?1 dry matter (DM)], Na (0.3 to 3 g kg^?1 DM) and Mg (1.0 to 2.4 g kg^?1 DM) concentrations were mostly below the required levels for ruminants. The lowest values were detected in February. K levels ranged between 6.6 g kg^?1 DM in February and 23.4 g kg^?1 DM in December; on average, K concentrations decreased about 10 g kg^?1 DM with advancing winter. Ca concentrations (2.9 to 7.4 g kg^?1 DM) hardly changed during winter. Related to higher growth rates of F. arundinacea before December, P and Ca concentrations were diluted, but regarding Mg, more wintergreen F. arundinacea reached higher values than L. perenne especially at the beginning of winter.     

Introgression of crown rust resistance from Festuca spp. into Lolium multiflorum

Crown rust, Puccinia coronata Corda, causes one of the most damaging foliar diseases in Italian ryegrass, Lolium multiflorum Lam. For introgression of crown rust resistance, highly resistant hybrids of the crosses Festuca arundinacea (2n = 6x = 42) ×L. multiflorum (2n = 4x = 28) and reciprocally, L. multiflorum (2n = 2x = 14) ×Festuca pratensis (2n = 4x = 28) and subsequently resistant recombinant individuals were used as female parents and susceptible cultivars of Italian ryegrass as male parents in three successive backcrosses. The BC₃ plants were selfed and crossed mutually. Uredospores of seven different crown rust isolates collected from plants of L. multiflorum, Lolium perenne, F. pratensis, F. arundinacea and L. multiflorum × F. pratensis hybrids were applied to identify the resistance or susceptibility of the parental species, backcrossed, selfed and intercrossed progenies. The various crown rust populations revealed a species-specific capability to infect plants of the Lolium-Festuca complex corresponding to the host species from which the spores originated. Selected BC⁹ plants, however, were found to be completely resistant to all crown rust populations tested. Successful introgression of the resistance was achieved from F. arundinacea as well as from F. pratensis. The resistance represents a dominant character, apparently based on a strong heterologous incompatibility between host and pathogen. In phenotype, bivalent formation during meiosis and in fertility, the novel germplasms are comparable with the L. multiflorum cultivars.     

Improved Drought Resistance in Introgression Lines Derived from Lolium multiflorum × Festuca arundinacea Hybrids

In a back-crossing programme involving Lolium multiflorum (the recurrent parent) and Festuca arundinacea, the diploid L. multiflorum phenotype was rapidly recovered with the inclusion, in some progeny, of a small number of genes from the fescue parent. In field drought trials derivatives of these backcross populations were on average less drought resistant than the L. multiflorum parental populations, but 3 % of individuals were as drought resistant as F. arundinacea. After only one cycle of selection and polycrossing these drought-resistant Lolium-like plants, the mean drought resistance of most progeny lines was significantly improved, in some cases to near that of F. arundinacea. Available evidence strongly indicates that this improved drought resistance was due to the transfer of genetic material from Festuca into Lolium. These populations will contribute to variety improvement and to our understanding of the genetics and physiology of drought resistance.     

Genomic structure of androgenic progeny of pentaploid hybrids, Festuca arundinacea × Lolium multiflorum

To assess the usefulness of the doubled haploid (DH) method in the breeding of forage grasses, a sample of anther-derived progeny of pentaploid F₁ hybrids of Festuca arundinacea × Lolium multifiorum was karyotyped using genomic in situ hybridization (GISH). The technique allowed scoring of the total number of chromosomes, the number of chromosomes contributed by each parent, and the number and positions of the Festuca-Lolium translocation breakpoints. Among 27 plants analysed, 13 belonged to three clones, effectively reducing the number of different progeny karyotyped to 17. These included 10 haploids, five doubled haploids and two plants for which the origins could not be explained. In all plants analysed, a mixture of chromosomes of both parents was present, including an average of 1.88 intergeneric translocations per plant. The translocation breakpoints were distributed along almost the entire length of the chromosome arms. Chromosome variation among androgenic progeny appeared much wider than that in the conventional backcross but low vigour and high mortality suggest that this additional variation may be difficult to exploit directly in breeding. However, a change in the pattern of recombination makes the entire genome accessible to manipulation.     

Estimation of introgression rate of a fescue isozymic marker into tetraploid Italian ryegrass at early generations of backcross

The potential for introgression of Festuca glaucescens into tetraploid ryegrass Lolium multiflorum was assessed using a simplex homeoallele at the phosphoglucoisomerase isoenzyme locus (Pgi-2). Overall, the F. glaucescens species-specific marker was found to be transmitted into Italian ryegrass at a rate close to 0.5 suggesting a high rate of homeologous pairing in BC1 and of intergeneric recombination in BC2. Significant variations in transmission rate among progenies in advanced generations indicated that recombination could result from various chromosome rearrangements possibly involving more than one fescue chromosome with some selection against duplex-like introgressed genotypes. Hence, introgressing F. glaucescens genes into tetraploid ryegrass appeared as further procedure to exploit versatility in fescue species, mostly restricted so far to introgression from F.arundinacea or F. pratensis into diploid ryegrass. Implications in terms of breeding strategy and marker-assisted selection are briefly discussed in respect to the genetic control of traits of interest to be introgressed. This revised version was published online in July 2006 with corrections to the Cover Date.     

Habitat and management affect genetic structure of Festuca pratensis but not Lolium multiflorum ecotype populations

Genetic diversity present in permanent grassland may be valuable for broadening gene pools in breeding programmes and for conservation of genetic resources. However, little is known about the amount of genetic diversity present at specific habitats and about site‐related factors affecting it. To identify valuable habitats, genetic diversity of 12 ecotype populations and four reference cultivars of both Festuca pratensis Huds. and Lolium multiflorum Lam. was analysed using Simple sequence repeat markers (SSR). Analysis of molecular variance revealed a larger within population variation for L. multiflorum (97.1%) than for F. pratensis (92.6%). F. pratensis ecotype populations were clearly separated from cultivars and formed three distinct subclusters according to the geographic regions they were sampled from. Differences between L. multiflorum ecotype populations and cultivars were small and no grouping of populations was observed. Thus, only F. pratensis ecotype populations were structured and habitat as well as management had a slight influence on genetic structure. This information may allow the design of individual strategies for targeted utilization of genetic resources in plant breeding programmes.     

Performance of Timothy-based Grass/Legume Mixtures in Cold Winter Region

This study sought to identify grass/legume mixtures that increase the yield and persistence of forage stands with improved nutritive quality in cold‐winter regions, compared with the standard mixture of timothy (Phleum pratense L.)/red clover (Trifolium pratense L.)/alsike clover (Trifolium hybridum L.). Timothy was mixed with either perennial ryegrass (Lolium perenne L.), meadow fescue (Festuca pratensis L.) or Kentucky bluegrass (Poa pratensis L.). The legumes in mixtures were red clover, alfalfa (Medicago sativa L.) or white clover (Trifolium repens L.). Averaged over three production years, the majority of mixtures had greater dry matter (DM) yields than the standard (8.35 t ha^?1). Timothy, grown alone and in three mixtures, outyielded the standard by 19–30 %. Yield reductions in mixtures over the 3‐year period were greatest with red clover, and least with bluegrass. Mixtures with alfalfa were highest in nitrogen (28.4 g kg^?1), while grasses grown alone (24.6 g kg^?1) and the standard mixture (25.1 g kg^?1) were the lowest in N. Mixtures with red clover or alfalfa had the least neutral detergent fibre (NDF), averaging 418 and 429 g kg^?1 respectively. Mixtures including white clover were initially low in NDF at 347 g kg^?1 in year 1 but increased to 550 g kg^?1 in year 3 as white clover composition declined in the sward.     

Infection of Lolium and Festuca spp. by Drechslera siccans and D. Catenaria

Summary Drechslera siccans was the commonest of the species of this leaf spot pathogen found on single spaced plants of Lolium perenne at Aberystwyth. D. catenaria and D. dictyoides were also frequently isolated. A wide range of genotypes within the host species L. perenne, L. multiflorum, Festuca arundinacea and F. pratensis, and structural hybrids between F. arundinacea and L. multiflorum, were all equally susceptible to conidial infection by D. siccans in the glasshouse and showed similar development of leaf spot symptoms, even though Festuca spp. are not normally attacked in the field. However, when exposed to infection out of doors there was significant variation in resistance, both between species and often also between genotypes within the same species. As in so many other instances of fungal infection, tetraploidy appeared to confer a higher level of resistance. The degree of resistance of a F. arundinacea × L. multiflorum amphiploid and two succeeding backcross generations to L. multiflorum approximated to that of the more susceptible L. multiflorum parent. By contrast to D. siccans, when plants were inoculated with D. catenaria in the glasshouse, both intervarietal and intravarietal variation in leaf spot development was evident. Resistance to D. siccans appears to depend partly on a limitation of conidial production, and to D. catenaria on restriction of hyphal growth following infection, which may explain the relative discrepancies between field and glasshouse results. The implications for resistance breeding are discussed.     

Cytogenetic Studies of Crosses between Lolium multiflorum Lam. and Festuca arundinacea Schreb. III. The Generations C1, C2 and C3

Meiotic observations of 56-chromosome plants of the C₁, C₂ and C₃ generations of the amphidiploid Lolium multiflorum × Festuca arundinacea showed that most of the chromosomes paired as bivalents. However, uni- and multivalents were frequently observed. No difference in meiotic regularity and stability could be detected between the different generations. The average chromosome number and; the percentage of 56-chromosome plants decreased with increasing generations. The direct use of the 56-chromosome amphidiploids of L. multiflorum and F. arundinacea in a breeding programme appears to be quite difficult, but these plants can be used in a backcross programme with Lolium and Festuca.     

Tall Fescue Adaptation to Low Nitrogen Fertilization in Relation to Germplasm Type and Endophyte Infection

Efficient crop growth with low nitrogen (N) application is becoming a requirement to face the concern on excessive N emission to the environment and the increasing cost of fertilizers. This study compared six natural populations and three improved varieties of tall fescue (Festuca arundinacea Schreb.) for dry‐matter yield over 4 years under ordinary N fertilization (450 kg ha^?1 in the 4 years) and very low N fertilization (only 40 kg ha^?1 prior to sowing). Both endophyte‐infected (EI) and endophyte‐free (EF) forms of each accession were sown. On average, ordinary fertilization implied 30 % higher yield than very low fertilization and the EI germplasm outyielded the corresponding EF germplasm (+4.4 %). Mean yield of improved varieties was higher than that of natural populations under ordinary fertilization (P < 0.05), whereas the two germplasm groups did not differ under very low fertilization. Accession × N fertilization interaction was found among natural populations but not among varieties (P < 0.05). A few natural populations were top‐yielding under very low fertilization and may be exploited, possibly in combination with endophyte infection, to select tall fescue varieties with enhanced performance under limited N application.